Our published experiments have indicated that the DNA demethylating agent 5 aza 2 deoxycytidine (DAC) mediates dramatic induction of CT-X genes such as NY-ESO-1, MAGE-A1, and MAGE-A3 in cultured lung cancer cells, but not normal human bronchial epithelia (NHBE). Furthermore we have demonstrated that the HDAC inhibitor Depsipeptide FK228 (DP) enhances DAC-mediated induction of CT-X genes in lung cancer cells. Induction of CT-X gene expression by sequential DAC/DP treatment coincided with a dramatic increase in expression of several BORIS splice variants in lung cancer cells. To date, the mechanisms mediating coordinate activation of CT-X genes and aberrant silencing of tumor suppressor genes in thoracic malignancies remain unclear. In recent studies, pyrosequencing and chromatin immunoprecipitation (ChIP) techniques were used to comprehensively examine chromatin alterations associated with repression/activation of NY-ESO-1, MAGE-A1, and MAGE-A3 in a panel of lung cancer lines exhibiting various CT-X gene expression profiles. Repression of CT-X genes in A549, Calu-6 and H841 cells as well as normal human bronchial epithelial cells coincides with CpG hypermethylation, recruitment of a variety of DNMTs, methyl CpG binding proteins, and histone demethylases including LSD1 and JARID1B, JARID1D, as well as deacetylation of histones H3/H4 and increased levels of H3K9me3 and H3K27me3 within the NY-ESO-1 promoter. In contrast, activation of NY-ESO-1 in untreated H1299 cells, or H841 cells following sequential DAC/DP exposure coincides with marked DNA demethylation, reduced levels of DNMTs, MBDs, and histone demethylases, accumulation of hyperacetylated core histones, as well as increased levels of H3K4me2, H3K4me3, and H3K9Ac within the NY-ESO-1 promoter. Knock-down of EZH2 or LSD1, JARID1B, JARID1D, or SIRT1 markedly enhanced DAC-mediated activation of NY-ESO-1. Comparable results were noted for MAGE-A1 and MAGE-A3 induction mediated by DAC. Additional studies have demonstrated that the polycomb inhibitor, DZNep also enhances DAC-mediated activation of CT-X gene expression in cancer cells, markedly enhancing recognition by allogeneic T cells expressing receptors for NY-ESO-1 and MAGE-A3. Furthermore, DZNep potentiates apoptosis mediated by DNA demethylating agents and HDAC inhibitors in lung cancer cells. Results of these studies have been presented at the recent annual meeting of the AACR, as well as an international cancer epigenetics symposium. A manuscript entitiled Inhibition of Histone Lysine Methylation Facilitates De-repression of Cancer-Testis Genes in Lung Cancer Cells: Implications for Adoptive immunotherapy of Cancer has been tentatively accepted for publication in Cancer Research pending minor revisions. Collectively these studies may facilitate the development of novel epigenetic strategies to induce apoptosis and augment immunogenicity of thoracic malignancies. A series of experiments have been undertaken to examine epigenomic alterations in malignant pleural mesothelioma (MPM) cells. Briefly, a panel of pleural mesothelioma lines and nomal mesothelial cell cultures initiated in our laboratory or obtained from commercial sources were processed for micro-array analysis, focusing on epigenetic modulators of gene expression. These experiments revealed aberrant expression of a variety of genes encoding DNA methyltransferases, histone acetyl transferases and istone Deacetylases in MPM cells. Interestingly, this analysis revealed over-expression of enhancer of zeste 2 (EZH2), suppressor of zeste 12 (SUZ12), and embryonic ectoderm development (EED), which encode key components the Polycomb repressive complex-2 (PRC-2) that mediates trimethylation of lysine 27 on histone H3 (H3K27Me3)- a repressive chromatin mark, which contributes to epigenetic silencing of tumor suppressor genes during malignant transformation. Quantitative RT-PCR (qRT-PCR), gel-based RT-PCR, and western blot experiments confirmed significant up-regulation of EZH2 and EED in MPM lines;EZH2 and EED over-expression coincided with increased H3K27Me3 levels in these cells. Additional qRT-PCR, RT-PCR, and immunohistochemistry experiments utilizing 20 primary MPMs and tissue micro-arrays containing 28 MPMs and 17 peritoneal mesotheliomas revealed over-expression of EZH2 in 85% of mesotheliomas compared to 12 normal mesothelia specimens. Knockdown of EZH2 or EED decreased global H3K27Me3, and diminished proliferation and migration of NCI-SB-MES1, NCI-SB-MES2, H28, and H2452 MPM cells (p less than 0.05). Furthermore, knockdown of EZH2 or EED decreased clonogenicity (p less than 0.05), and tumorigenicity (p less than 0.05) of NCI-SB-MES1;the effects of EED knockdown were more pronounced than EZH2 inhibition in these cells. DZNep, a novel inhibitor of polycomb expression, mediated dose-dependent depletion of EZH2, EED, and H3K27Me3, and inhibited proliferation (p less than 0.05), migration (p less than 0.05), clonogenicity (p less than 0.05), and tumorigenicity (p less than 0.05) of MPM cells. Additional experiments revealed that the growth inhibition coincided with up-regulation of RASSF1A, a tumor suppressor and known polycomb target that is aberrantly silenced in MPM. Collectively, these data demonstrate that aberrant expression of PRC-2 contributes to the malignant phenotype of MPM, and suggest that PRC-2 may be a novel target for mesothelioma therapy. Results of these studies were presented in oral formal at the recent International Mesothelioma Interest Group Meeting in Kyoto, Japan. A manuscript pertaining to these experiments is in preparation.